Musharova Olga, Klimuk Evgeny, Datsenko Kirill A, Metlitskaya Anastasia, Logacheva Maria, Semenova Ekaterina, Severinov Konstantin, Savitskaya Ekaterina
Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.
Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.
Nucleic Acids Res. 2017 Apr 7;45(6):3297-3307. doi: 10.1093/nar/gkx097.
During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
在引发的CRISPR适应过程中，间隔序列优先从CRISPR干扰机制识别的DNA中选择，就I型CRISPR-Cas系统而言，该机制由结合了效应器Cascade复合物的CRISPR RNA（crRNA）组成，Cascade复合物定位互补靶标，以及Cas3执行核酸酶/解旋酶。Cas1和Cas2蛋白复合物能够在CRISPR阵列中插入新的间隔序列。在此，我们表明，在经历引发适应的大肠杆菌细胞中，外源DNA的间隔序列大小的片段与Cas1相关联。基于对核酸酶消化的敏感性，相关的DNA不是处于标准双链状态。间隔序列大小的片段以依赖于Cas1和Cas3的方式从外源DNA的一条链上切割下来。这些片段是从更长的对S1核酸酶敏感的外源DNA片段产生的，而这些更长的片段需要Cas3来产生。我们提出，在CRISPR干扰过程中，Cas3产生外源DNA片段，这些片段被Cas1-Cas2适应复合物识别，该复合物切除间隔序列大小的片段并将它们引导至插入CRISPR阵列。
